Transcriptional regulatory mechanisms determine cellular phenotypes by directing precise patterns of temporal gene expression. The central hypothesis underlying this proposal is that one or more key testis- specific transcription factors mediate the signal for terminal differentiation of male germ cells by activation of spermatid-specific genes. The goal of this research program is to isolate and characterize testicular transcription factor(s) involved in postmeiotic gene regulation. The study focuses on a testis-specific gene encoding the acrosomal protein SP-10, a model gene for studying regulation of post- meiotic, testis-specific gene expression. This gene 1) Exhibits a temporal pattern of gene expression, being restricted to the golgi and cap phases of spermiogenesis; 2) Is well characterized in both human and mouse; and 3) Is expressed uniquely in round spermatids, and is present as a single copy gene. Evidence indicates that SP-10 is not under the transcriptional control of CREM, the only well characterized transcription factor in testis. The current proposal utilizes the SP-10 gene promoter to capture novel testis-specific transcription factors and to test the hypotheses that such factors arise in a stage-specific manner during spermatogenesis to bring about coordinate expression of spermatid-specific genes. To identify and characterize the transcription factors which regulate SP-10 gene expression; transgenic mice will be generated using SP-10 promoter +GFP reporter constructs, and gel shift assays and Dnase I footprinting will be performed to identify the cis-acting elements. To determine the functional role of the putative transcription factor(s) in SP-10 gene expression, in vitro transcription assay will be developed using testicular nuclear extracts and SP-10 promoter driven G-less cassette. 3) To test the hypothesis that SP-10 transcription factor(s) appear in a developmental-stage specific manner in the testis, in situ hybridization of testis sections, Northern analysis and RT-PCR of testicular RNA obtained from prepuberal as well as adult mice will be performed. Identification of the transcription factor(s) governing SP-10 gene expression will pave the way for future studies concerning the role of these factor(s) in coordinate gene regulation during spermiogenesis. Achievement of these aims will add significantly to the knowledge of male germ cell differentiation at the molecular level. The proposed studies are clinically relevant because meiotic arrest in spermatogenesis is a documented cause of infertility in men. The factors governing gene regulation of spermiogenesis may provide new approaches to male contraception.